Project Summary/Abstract The goal of this project is to develop a highly rigorous technology platform utilizing lichen and un- sequenced fungi to revive the discovery pipeline for fungal-derived therapeutics capable of treating chronic infections and health conditions. The emergence of drug resistant microbes, the diminishing supply of novel classes of antibiotics, and the dramatic reduction in discovery and development of natural products (NPs, also termed secondary metabolites, SMs) and other small molecule compounds from bacteria and fungi for anti-infective, anti-proliferation and anti- inflammation agents since 1960s have amplified a serious public health concern championed by the CDC and WHO. We posit that the revival of large-scale drug discovery pipelines using under- exploited microbes including lichen fungal symbionts and un-sequenced fungi will provide a new cadre of novel drug leads and solutions towards the antibiotic-resistance crisis. Fungal SMs are also important sources of anticancer compounds among other widespread clinical uses. However, only 1% or less of filamentous fungi have been sequenced and high throughput sequencing has shown that only about 10% of fungal SM-biosynthetic gene clusters (BGCs) are expressed under laboratory conditions. Therefore, revolutionary technologies and tools are urgently needed to discover and more effectively dissect the biosynthesis of fungal SMs in order to more efficiently access novel fungal metabolites as potential pharmaceutical agents. Recently, we developed a novel fungal artificial chromosome/mass spectrometry (FAC-MS) method that allows the direct capture, heterologous expression and chemical analysis of an entire set of large intact SM-BGCs from sequenced fungi as shown in Clevenger et al., Nat. Chem. Biol, 2017. We have also shown that shuttle bacterial artificial chromosome (BAC) technology combined with BAC pooling, indexing, and next-gen sequencing (NGS) can achieve 100kb-linked sequencing and assembly. Therefore, in this project, we will develop an innovative fungal technology platform by integrating FAC-MS with BAC/NGS sequencing and bioactivity profiling to prove the concept that novel bioactive SMs can be captured from hard to grow (e.g. lichen fungal symbionts, Phase I) and un-sequenced fungi (Phase II). This technology should improve fungal SM discovery 100~1000 fold and result in the discovery of at least 5 novel antimicrobial drug leads from lichen and un-sequenced fungi.